APPROACHES TO SYNTHESIS OF ([1,2,4]TRIAZOLO[1,5- c ]QUINAZOLIN-2-YL)BENZOIC ACIDS AS POTENTIAL ANTI-INFLAMMATORS

regularities of of protons anti-inflammatory and the quinazoline heterocycle. aim of the present study is to develop methods for the synthesis of [1,2,4]triazolo[1,5- c ]quinazolin-2-yl)benzoic acids as potential anti-inflammatory agents. Quinazolin-4(3 H )-ylidene)hydrazides (hydrazones) of benzenedicarboxylic acids, their esters, products of their heterocyclization and nucleophilic degradation were the subjects of the study. The structure of the synthesized compounds was confirmed by elemental analysis and NMR spectroscopy. Anti-inflammatory activity was studied in a model of acute aseptic inflammation («carrageenan test») in rats. Possibilities and limitations of synthesis of [1,2,4]triazolo[1,5- c ]quinazolin-2-yl)benzoic acids and

Non-steroidal anti-inflammatory drugs (NSAIDs) are a common and diverse group of compounds that are mainly used to treat patients suffering from pain and inflammation (chronic pain, osteoarthritis, rheumatoid arthritis, postoperative surgical conditions, etc.) [1,2]. In addition, this group of drugs is used as analgesics and antipyretics. Aryl carboxylic acids and their derivatives, which were among the first to be introduced into medical practice, are still important among NSAIDs [3]. In addition to the well-known salicylic and acetylsalicylic acids, this group of anti-inflammatory agents includes salts of salicylic acid, salsalate and diflunisal. Recently, representatives of anthranilic (2-aminobenzoic) acid derivatives have also been widely used, namely fenamates (mefenamic, meclofenamic, flufenamic and tolfenamic acids).
Despite the interclass chemical diversity of NSAIDs, their pharmacological action is based on the inhibition of cyclooxygenase (COX)/prostaglandin-endoperoxide synthase (PGHS-1 and PGHS-2), of regulatory enzymes involved in the biosynthesis of prostaglandins (PG) -mediators of inflammation. However, due to non-selective inhibition of COX-1 and COX-2, the group of NSAIDs, despite its high efficiency, has significant side effects (complications of the gastrointestinal tract, cardiovascular system, liver, kidneys, etc.) [4]. With this in mind, medical chemists still pay considerable attention to the design and synthesis of NSAIDs [5]. Undoubtedly, these studies are devoted firstly to the modification of the carboxyl group to esters; secondly, its «bioisosteric» replacement by other structural fragments; thirdly, the search for new compounds with anti-inflammatory activity among heterocyclic compounds. These modifications among NSAIDs are carried out to increase their selectivity for biological targets, search for compounds that would affect other biological targets (phospholipase, lipoxygenase), solubility, redox processes, etc. [5]. Given the above facts, the creation of hybrid molecules that would combine in one structure a fragment with antiinflammatory activity (phenylcarboxyl group) and a quinazoline heterocycle, which is also characterized by this activity [6][7][8], is a promising and relevant direction. Moreover, molecular pharmacologists have explained the key role of the «pharmacophore» carboxyl group in the formation of enzyme-ligand interactions and the effect of its presence on activity and selectivity [9][10][11][12].
Ethyl 3- [1,2,4]triazolo [1,5-c]quinazolin-2-yl)benzoate (3.1  Method for the synthesis of (quinazolin-4(3H)-ylidene)hydrazineylidene)methyl) benzoic acids (5). To a suspension of 1.6 g (0.01 M) of 4-hydrazinoquinazoline (1.1) in 10 ml of dioxane 0.11 M of the corresponding formylbenzoic acid was add. The suspension was refluxed for 1.5-2 hours, cooled and poured into water. The formed precipitates were filtered and dried. If necessary, crystallized from a mixture of DMF-water (10:1).  (5.3 The general method for the synthesis of [1,2,4]triazolo [1,5-c]quinazolin-2-yl)benzoic acids (6). 1.46 g (5 mmol) of the corresponding quinazolin-4(3H-ylidene)hydrazinylidene) methyl)benzoic acids (5) and 1.23 g (15 mmol) of anhydrous sodium acetate were added to 40 ml of glacial acetic acid with constant stirring. A solution of 0.8 g of bromine (5 mmol) in 10 ml of glacial acetic acid was then added dropwise to the starting compounds. Continue stirring for 3 hours, after which the mixture was poured into cold water. The formed precipitate was filtered off and dried. Crystallized from ethanol or dioxane. Anti-inflammatory activity. Evaluation of anti-inflammatory activity of the synthesized compounds was conducted on 84 Wistar white rats (weight 150-160 g), obtained from the nursery «Institute of Pharmacology and Toxicology of Ukraine» (Kyiv). All experimental procedures and treatment were carried out according to the European Convention and «Regulations on the use of animals in biomedical research» [15]. Screening of the synthesized compounds with estimated anti-inflammatory activity began with the study of their effect on exudative phase of acute aseptic inflammation («сarrageenan» test) [16]. Phlogogen (1% aqueous solution of λ-сarrageenan) was subplantally injected in a dose of 0.1 ml in the rats' hind right paw. The left one was used as a control. Intragastric administration of the studied compounds was conducted using atraumatic probe as water solution or finely dispersed suspension stabilized by Tween-80 in a dose of 10 mg/kg 1 hour before the injection of phlogogen. The reference drug Diclofenac sodium was administered intragastrically in a recommended dose of 8 mg/kg for pre-clinical studies. Measurement of paws volume was conducted before the experiment and in 4 («сarrageenan» test) hours after injection of phlogogen using the described methods. The activity of these substances was determined by their ability to reduce the swelling compared with control group and was expressed in percentage. It showed how the substance inhibited phlogogen swelling in relation to control swelling where the value was taken as 100%. The activity of the studied compounds was calculated as following:

4-(Quinazolin-4(3H)-ylidene)hydrazineylidene)methyl)benzoic acid
, where A -antiexudative activity, %; Vpe -the volume of paw edema in the experiment; Vhe -the volume of healthy paw in the experiment; Vpc -the volume of paw edema in control; Vhc -the volume of healthy paw in control.
Statistical data processing was performed using a license program «STATISTICA® for Windows 10.0» (StatSoftInc., № AXXR712D833214FAN5) and «SPSS 16.0», «Microsoft Office Excel 360». The results were presented as mean ± standard error of the mean. Arithmetic mean and standard error of the mean were calculated for each of the studied parameters. During verification of statistical hypothesis, null hypothesis was declined if statistical criterion was p < 0.05 [17].

R e s u l t s a n d d i s c u s s i o n
We have previously shown that the interaction of 4-hydrazinoquinazoline (1.1) with anhydrides alkanedicarboxylic acids under extreme conditions leads to the formation of the corresponding ([1,2,4]triazolo[1,5-c]quinazolin-2-yl)alkane-carboxylic acids [18]. An attempt to synthesize these heterocycles with cycloalkane(aryl-)carboxyl groups at 2 nd position using aromatic or alicyclic anhydrides (endic, phthalic anhydride and its hydrogenated analogues) in this reaction leads to the formation of the corresponding cyclic 4-imidoaminoquinazolines [19].
To study the possibility of the heterocyclization in another direction, namely with the formation of triazoloquinazoline systems with a carboxyl group, considering the purpose of the work, we have developed a number of original approaches. The first approach was to form monoesters of (2-(quinazolin-4(3H)-ylidene)hydrazine-1-carbonyl)benzoic acids (2) by interreacting of 4-hydrazinoquinazoline (1.1) with benzenedicarboxylic acid monoesters in the carbonyldiimidazole synthesis (Fig. 1).
Another approach to the formation of triazolo[c]quinazoline systems is the oxidative heterocyclization of 4-(benzylidene)hydrazineylidene)-3,4-dihydro-quinazolines [21]. The method we have used showed that treatment of compounds (5) with bromine in glacial acetic acid leads to the formation of [1,2,4]triazolo [1,5-c]quinazolin-2-yl)benzoic acids (6.1, 6.2, Fig. 1). The probable mechanism of this reaction is realized through the stage of electrophilic addition of bromine on the azomethine fragment, formation of carbocation with subsequent nucleophilic attack of the endocyclic atom of the Nitrogen cycle with the formation of s-triazolo [4,3-c]quinazolines. It is important, that s-triazolo [4,3-c]quinazolines were isomerized to the corresponding [1,5-c]-series under these conditions. However, as in the above case, compound (5.1) forms a mixture of products, which is also, in our opinion, complicated by Dimroth rearrangement (Fig. 2).
Structure and individuality of synthesized compounds was confirmed by elemental analysis, chromato-mass and 1 H NMR spectrometric. A quasimolecular ion [M+1] was registered in the chromato-mass spectra substances, which confirms their structure and individuality. 1 H NMR spectra also indicate their unambiguous formation. Thus, the 1 H NMR spectra of compounds ((2) were characterized by signals of exchange singlet protons of the endocyclic 3-NH-and -NNHCO-groups at 12.05-11.80 ppm and 11.12-10.83 ppm, respectively. The aromatic protons of the quinazoline ring and the phenyl substituent of compounds (2) were in most cases manifested in the form of multiplet signals, which were associated with tautomeric transitions in the molecule (hydrazide-hydrazone tautomerism). Signals with characteristic splitting and chemical shift were manifested in the strong magnetic field of the spectra of compounds (2) [22]. Exchangeable protons of -COOH and protons of endocyclic 3-NH groups were observed at 13.25-12.71 ppm and 11.76-11.61 ppm, respectively in 1 H NMR spectra of compounds (5). The characteristic azomethine proton (-N=CH-) and the proton of 2 nd position of the heterocycle of compounds (5) resonate as singlets at 9.19-8.48 ppm and 8.61-7.89 ppm, respectively. Aromatic protons have «classical» chemical shifts and multiplicity, which is characteristic for these systems [18,22].
In favor of the heterocyclization and oxidative cyclization of compounds (2) and (5), unambiguously indicate the 1 H NMR spectra of compounds (3) and (6). Thus, the signals of singlet protons at the 5 th position of the heterocycle in these compounds was characteristic and resonates at 9.68-9.63 ppm, indicating the course of Dimroth rearrangements [18]. Regarding the reaction of nucleophilic degradation of the pyrimidine cycle of compounds (3), the uniqueness of its course indicates the absence in compounds (4) of the proton at the 5 th position and the appearance of broad one-proton singlet protons of the triazole cycle at 14.46-14.30 ppm and NH 2 groups at 6.71 ppm [24]. The latter is not registered in compound (4.2), due to tautomerism and interaction with the COOH group of the molecule. Additionally, this confirms the 13 C NMR spectrum of compound (4.2), in which the required number of chemical shifts of the carbon atom was observed. Studies on anti-inflammatory activity have shown that (quinazoline-4(3H)-ylidene) hydrazides of dicarboxylic acids (2) inhibit the development of carrageenan edema by 3,44-32,29% compared to control, inferior to diclofenac sodium (AA = 69,79%, tablе). In this case, there is a certain dependence, namely the decrease in activity in the series p > o > m-position of the ester group. The corresponding hydrazones (5)   The formation of planar [1,2,4]triazolo [1,5-c]quinazoline cycle (3) leads to loss of antiinflammatory activity of the corresponding esters (AA = 16,35-20,67%). Importantly, higher anti-inflammatory activity was observed for compound (3.1), in which the ester group is in the m-position. Compound (6.1) with a carboxyl group in the m-position (AA = 37,15%) turned to be also the most active, that is speaking about [1,2,4]triazolo [1,5-c]quinazolin-2yl)benzoic acids (6). Replacing of the substituent to the p-position leads to the loss of activity (AA = 23,96%). Nucleophilic degradation of the triazolo[1,5-c]quinazoline cycle showed that ethyl 4-(5-(2-aminophenyl)-1H-1,2,4-triazol-3-yl)benzoate (4.1) was more active (АА = 27,78%). However, the corresponding acid was an inactive compound (AA = 15,06%).
2. 1 H NMR spectra were studied and regularities of splitting of characteristic protons in synthesized compounds were established.
Funding. This research was a part of the scientific project «Directed search for biologically active substances among annulated quinazoline and pteridine derivatives» (problem «Pharmacy», state registration №0117U 006961, period of study 2017-2022).